Endonuclease-enhanced helicase-dependent amplification

ABSTRACT

The invention provides methods and compositions for enhancing the speed and sensitivity of helicase-dependent amplification through the use of an endonuclease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/146,602, filed Oct. 7, 2011, now allowed, which is a National Stagefiling under 35 U.S.C. §371 of PCT/US2010/023089, filed Feb. 3, 2010,and Publication No. WO/2010/091111, published Aug. 12, 2010, whichclaims the benefit of U.S. Application No. 61/149,390, filed Feb. 3,2009.

CROSS-REFERENCE TO A SEQUENCE LISTING

A Sequence Listing is being submitted electronically via EFS in the formof a text file, created Mar. 3, 2014, and named seqlist0418960824.txt(732 bytes), the contents of which are incorporated herein by referencein their entirety.

FIELD

The present invention relates to methods and compositions to improve thespeed and sensitivity of helicase-dependent amplification.

BACKGROUND

Nucleic acid amplification has been widely used for research andmolecular diagnostics. PCR (polymerase chain reaction) is a traditionalmethod for nucleic acid amplification which requires themiocycling. Heatdenaturation during PCR themiocycling is a powerful and important stepto uniformly separate DNA duplexes, generating single-stranded templatesfor subsequent primer annealing. Several platforms exist for isothermalamplification, including SDA (strand displacement amplification), TMA(transcription-mediated amplification), RCA (rolling cycleamplification), LAMP (loop-mediated amplification), NASBA (nucleic acidsequence-based amplification), and HDA (helicase-dependentamplification). HDA is different from other isothermal technologiesbecause the HDA technology employs a helicase to separate thedouble-stranded nucleic acid. It is desirable to improve the speed andsensitivity of nucleic acid amplification technologies with high speedand high sensitivity, particularly for use in research and diagnosticapplications.

Several methods have been developed to improve speed and sensitivity forPCR. Some of the methods that can improve PCR performance have also beensuccessful for improving HDA performance. However, HDA is different fromPCR, as HDA can be performed isothermally and relies on a helicase toseparate the strands of double-stranded nucleic acids. Several specificmethods have been applied to improve HDA. For example, previous studieshave found that the protein concentrations of DNA polymerase andhelicase can be optimized to improve the performance of HDA. Althoughincreasing amounts of enzymes (helicases and DNA polymerases) canincrease detection speed, the Limit-of-Detection (LoD), the minimumnumber of target nucleic acids required for reliable detection,generally remains unchanged at, for example, 5-50 copies.

HDA uses helicase(s) to separate a DNA duplex. Helicase is not asequence-specific protein and therefore does not specifically recognizea target region. The efficiency of the helicase separating thedouble-stranded nucleic acid, especially in the target region specifiedby forward and reverse primers, is therefore a rate-limiting factor.Based on the unwinding direction, helicases can be grouped into twomajor types: the 3′-5′ helicases and the 5′-3′ helicases. Thewell-studied E. coli UvrD helicase unwinds DNA in a 3′ to 5′ direction(Matson, S. W. J. Biol. Chem., 261, 10169-10175. (1986)). Some helicasescan unwind duplex nucleic acid from blunt ends; advantages of using suchhelicases are described in U.S. Pat. No. 7,282,328. Other helicasesrequire a single-stranded “tail” on the substrate duplex, generally a 3′tail for 3′-5′ helicases and a 5′ tail for the 5′-3′ enzymes.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that the speedand sensitivity of helicase-dependent amplification can be improvedthrough the use of an endonuclease, such as a restriction endonuclease.The extraction and purification of nucleic acid substrates typicallyshears or nicks them, generating randomly-distributed accessible endsfor a helicase to load onto the duplex. Perhaps as a result, the art hasnot previously appreciated any need for or benefit from introducingadditional free ends in a target for helicase-dependent amplification.Surprisingly, the inventors have now discovered that treating the targetnucleic acid with an endonuclease can significantly improve both thesensitivity and speed of helicase-dependent amplification.

Accordingly, in one aspect the invention provides an improvedcomposition for helicase-dependent amplification. The compositionincludes a helicase, a DNA polymerase, at least one primer, and anendonuclease. The endonuclease can be a restriction endonuclease, suchas a Type II or Type IIs restriction endonuclease. Exemplaryendonucleases include AvaII, BanII, AseI, SspI, SpeI, MboI, MboII, SwaI,BsrI, BtsCI, CviQI, CviAII, Tsp45I, Fnu4HI, Ms1I, MspA1I, SacII, AgeI,DraI, and XmnI. The helicase is preferably thermostable. The compositioncan include additional components, such as deoxynucleotides, buffer,magnesium, sodium, one or more additional primers, and/or singlestranded binding proteins, for example.

The invention also provides methods for assaying a sample by combining acomposition like those described above with a sample containing orpotentially containing a nucleic acid that hybridizes to one or moreprimers in the composition. In this way, the presence or absence of thetarget nucleic acid can be detected, the amount of a target nucleic acidcan be measured, or a target nucleic acid known to be present can beamplified to facilitate its subsequent characterization (e.g.determining the presence or absence of a genetic variation) or otheruse. For example, the sample can be a biological sample from a subjectand can include human tissue, cells or body fluid (e.g. blood, serum,plasma, lymph, urine, sweat, tears, semen, vaginal fluid, nippleaspirate, sputum, ejaculate, saliva, bronchial lavage, pleural effusion,peritoneal fluid, amniotic fluid, glandular fluid, fine needleaspirates, spinal fluid, conjunctival fluid, or cerebrospinal fluid),and the target nucleic acid can be a human nucleic acid or a foreignnucleic acid (e.g. from a virus, bacterium, fungus, parasite, etc.).Alternatively, the sample could be an environmental, animal orindustrial sample. In one embodiment, the sample contains no more than 1ng of DNA (e.g. no more than 0.1 ng or no more than 0.01 ng). In someembodiments, the sample contains no more than about 300 copies of thetarget nucleic acid (e.g. no more than about 30 copies or no more thanabout 3 copies.).

The invention also provides methods for amplifying a nucleic acid bytreating the nucleic acid with an endonuclease and amplifying thetreated nucleic acid in a helicase-dependent amplification reaction. Theamplification can follow the treatment step, or the two processes can beconcurrent. The endonuclease can be a restriction endonuclease such as aType II or a Type IIs restriction endonuclease. Exemplary endonucleasesinclude AvaII, BanII, AscI, SspI, SpeI, MboI, MboII, SwaI, BsrI, BtsCI,CviQI, CviAII, Tsp451, Fnu4HI, Ms1I, MspA1I, SacII, AgeI, DraI, andXmnI. In one embodiment, the helicase-dependent amplification reactionincludes a helicase (preferably thermostable), a DNA polymerase, abuffer, deoxynucleotides, magnesium, and at least one primer.

In each of the various methods of practicing the invention, theendonuclease preferably, but not necessarily, cleaves the nucleic acidwithin 5 kb (and perhaps within 500 bp) of the nucleotide sequence inthe nucleic acid that hybridizes to the primer (i.e. the primer bindingsite).

Additional aspects and advantages of the invention will be apparent fromthe following description of certain particular embodiments of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 and FIG. 2 are graphs showing the effects of restrictionendonuclease SspI on helicase-dependent amplification from 50,000 copiesof plasmid DNA (FIG. 1) or from 20 ng human genomic DNA (FIG. 2) for aone hour amplification at 63.5° C. FIG. 1 shows amplification from50,000 copies plasmid DNA. Ct (the threshold cycle, defined as the cyclenumber at which the fluorescence level has exceeded the detectionthreshold) is 14 for the assays without SspI, but is reduced to around11.5 for the assays with SspI. FIG. 2 shows amplification from 20 nghuman genomic DNA; Ct is around 14 for the assays with or without SspI.

FIG. 3 and FIG. 4 are graphs showing the effects of restrictionendonucleases SpeI (FIG. 3) and BanII (FIG. 4) (generating 5′ or 3′ endsafter cleavage, respectively) on helicase-dependent amplification from50,000 copies of plasmid DNA with a one hour amplification at 63.5° C.FIG. 3 shows the effects of SpeI. Ct is 14 for the assays without SspI,and is reduces to around 11.5 for the assays with SspI. FIG. 4 shows theeffects of BanII: Ct is 13 for the assays without BanII, and is reducedto about 11 with BanII.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

The invention improves the sensitivity of, and accelerates,helicase-dependent amplification through the use of endonucleases.Restriction endonucleases, nicking endonucleases, and homingendonucleases recognize and cleave specific nucleic acid sequences,generating 5′ss ends, 3′ss ends or blunt ends. More than 3,000restriction endonucleases with over two hundred different specificitieshave been isolated from bacteria (Roberts and Macelis, Nucleic AcidsRes. 26:338-350 (1998)). Type II restriction endonucleases are the mostcommonly used restriction enzymes for molecular biology applications:they cut DNA at defined positions close to or within their recognitionsequences. They produce discrete restriction fragments and distinct gelbanding patterns and are the only class of restriction enzyme commonlyused in the laboratory for DNA analysis and gene cloning. Examples oftype II enzymes include EcoRI, HindIII, SspI, and BanII, each cleavingDNA within its recognition sequence. The next most common Type IIenzymes, usually referred to as “type IIs,” are those such as FokI andA1wI that cleave outside of their recognition sequence to one side.During an HDA reaction, the selected restriction endonucleases generatebreak points (or ends) of the nucleic acid substrates that are near atarget region. These resulting ends recruit helicases to the adjacenttarget region and thus increase the chance or frequency of the helicaseunwinding a target region.

In an embodiment, an endonuclease is used prior to an HDA reaction(separate from the HDA reaction, in a two-step format). In such atwo-step format, restriction endonucleases digest the DNA targets firstat the optimal working temperature, typically in an HDA reaction bufferor in a buffer that does not inhibit the following HDA reaction. Thedigested nucleic acid substrates can then be used in the subsequent HDAreaction. In another embodiment, the endonuclease is used together withan HDA enzyme mix (in a one-step format, generally including a helicase,a DNA polymerase, and optionally single-stranded DNA binding proteins).In such a one-step format, the reaction speed of the endonucleasesshould be fast enough to generate a certain number of specific endsduring the time of reaction set-up (including, if applicable, the timeof adding the master reaction mix to each sample in the individual tube,mixing, and bringing the sample to an incubator which has already beenset to the HDA reaction temperature). In general, the set-up time may bein the range of about 2 to about 15 minutes, depending on how manysamples are to be tested. As an illustrative example, the “time-saverqualified enzymes” from New England BioLabs Inc, (NEB, Ipswich, Mass.)provide restriction endonucleases with fast reaction speeds. This groupof fast endonucleases digest about 1 μg of DNA in about 5 minutes usingabout 1 μl of enzyme under recommended conditions. The one-step formatis easier to perform as there is no additional step required to add therestriction endonucleases. On the other hand, fewer restrictionendonucleases are as suitable for the one-step format because itrequires fast speed and high digestion efficiency. In general, an enzymethat is suitable for the one-step format can always be used in atwo-step reaction.

Several preferred restriction endonucleases for use in the presentinvention arc selected on the basis of one or more the followingfactor(s):

-   -   Fast reaction speed. If the endonucleases are used together with        an HDA enzyme mix, reaction speed is an important factor in the        improvement in amplification. The “time-saver qualified enzymes”        from NEB are good choices, for example. However, if the        endonucleases are used separately in a two-step reaction,        reaction speed need not be an issue.    -   The buffer for endonuclease cleavage is preferably compatible        with HDA reaction buffer: the enzyme's activity is not inhibited        by the reaction buffer used for HDA.    -   The endonuclease does not or is not known to recognize, bind or        cleave within the target sequence to be amplified. If the target        sequence is cleaved by a restriction enzyme, the truncated        target region can no longer be used as a template for        amplification.    -   The endonuclease does not recognize, bind or cut the        corresponding internal control sequence, if an internal control        is included in the HDA reaction.    -   Proximity of the cleavage site to the target region. Ideally,        the cleavage site can be within 5,000 bp of the target region.        More preferably, the cleavage site is only 1-500 bp away from        the target region.    -   To improve reaction performance without cleaving the target        sequence, the sequence in the region for restriction enzyme        digestion and HDA amplification should be relatively conserved.        Highly polymorphic regions of a target nucleic acid are        preferably avoided.    -   The cleavage frequency of an endonuclease is determined, in        part, by the number of base pairs it recognizes. Some        restriction endonucleases recognize 6 base pairs. For example,        the restriction endonuclease SspI recognizes AATATT and cleaves        in the middle. As another example, restriction endonuclease SpeI        recognizes ACTAGT and cleaves between A and C. In general,        restriction endonucleases that recognize longer sequences (less        frequent cutters such as SspI and SpeI) show more enhancement in        most of the HDA reactions. The high-frequency cutters (for        example, those that recognize 4 base pairs) will not only        generate restriction ends close to the targets, but also        generate more ends in the entire nucleic acid substrates. Since        all ends will recruit helicases, higher numbers of available        ends in the nucleic acid substrates may compete with ends that        are adjacent to the specific target sequence. This “dilution”        effect can reduce the effectiveness of the restriction enzyme in        enhancing HDA.

In an embodiment, the restriction endonucleases are enzymes which digestthe DNA at a lower temperature during reaction setup (for example, fromabout 0° C. to about 37° C.).

In an embodiment, the improvement effects of the endonucleases depend onthe amount of DNA. In a sample having very low amounts of the initialtargets, for example 1-50 copies of DNA (for example, plasmid DNA,bacteria or virus genomic DNA, or human genomic DNA), endonucleasecleavage can greatly improve the chance of helicases binding to thelimited number of targets, and make the reaction more consistent at lowtarget concentrations. When an HDA reaction with low-initial-target isevaluated in real-time, the Ct (the threshold cycle, defined as thecycle number at which the fluorescence level has exceeded the detectionthreshold) is reduced if an endonuclease is used. This indicates thatthe HDA speed is enhanced by the restriction enzyme, evidenced by theearlier appearance of the fluorescent signal across the Ct line. Inaddition, the success rate of the HDA reaction with low-initial-targetis improved by using appropriate endonucleases: the standard deviationof Ct is reduced. Restriction endonucleases also reduce primer dimerformation (caused by non-specific primer annealing and amplification) atlow target concentrations. In a sample having a medium amount of theinput targets, for example, more than 50 copies of DNA, endonucleasecleavage still improves the detection speed. If the experiment isevaluated by real-time assay, the time needed to see a positivefluorescent signal across the Ct line is reduced by about 2 to about 6minutes. On the other hand, in HDA reactions with very high amounts ofinput substrate, for example, about 20 ng of human genomic DNA, therewill be an overwhelming number of non-specific ends, exceeding thenumber of helicases. In this case, no significant enhancement effects bythe restriction endonucleases are observed.

In an embodiment, a mixture of one or more compatible endonucleases foruse with the corresponding HDA can be selected. For example, anendonuclease which can cleave the upstream sequence of the target can becombined with an endonuclease which can cleave the downstream sequenceof the target.

In an embodiment, the use of restriction endonucleases in an HDAreaction is compatible with other methods to improve HDA performance.For example, the methods include, but are not limited to, proteinengineering or modification to improve the function of the protein(helicases, polymerases, other accessory proteins).

The following experimental examples are provided to illustrate certainembodiments of the invention, but are not intended to limit theinvention.

EXAMPLES Example 1 Use of Endonucleases to Improve Reaction Speed of HDA

In this example, a method is disclosed to improve HDA reaction speed byrestriction endonuclease cleavage. The target of amplification waswithin Homo sapiens breast cancer 1 (Genbank accession No. NT 010755REGION: 4967748 . . . 4971174, 3427 bp). This region was cloned intopCR-Blunt II-TOPO (Invitrogen, Carlsbad, Calif.), named as pBRCA-TOPO.Specific primers BRCA2F (AGCTTAGCAGGAGTCCTAGCCCTTT) (SEQ ID NO: 1) andBRCA2R (TCTGAGGACTCTAATTTCTTGGCCC) (SEQ ID NO: 2), were used to amplifyan 85 bp fragment from this region. In order to compare the effects ofendonuclease cleavage from a medium amount of DNA and a high amount ofDNA (with overwhelming numbers of ends), the same endonuclease wastested with different targets: plasmid DNA (50,000 copies of pBRCA-TOPO,around 0.38 pg) vs. human genomic DNA (20 ng, around 6000 copies). 20 ngof human genomic DNA contain many more ends than 0.38 pg of plasmid DNA.

The restriction endonuclease SspI (New England BioLabs, Inc, Ipswich,Mass.) was chosen to show the effects of enzyme cleavage, which cancleave the sequence around 187 bp downstream of the specificamplification region. The target sequence of the enzyme is AA^(↓)ATT.After cleavage, blunt ends are generated. The HDA reaction was performedfirst by combining the following components to produce a reaction mastermix with a final volume of 45μ/reaction (make 4× reaction master mix for2 tested samples):

-   -   5 μl 10× Annealing buffer II (see below*)    -   0.75 μl 5.0 μM BRCA2F primer (SEQ ID NO: 1)    -   0.75 μl 5.0 μM BRCA2R primer (SEQ ID NO: 2)    -   2.0 μl 100 mM MgSO₄    -   2.0 μl 500 mM NaCl    -   2.0 μl 10 mM dNTP    -   1.5 μl 100 mM dATP    -   0.5 μl EvaGreen (20×, Biotium, Hayward, Calif.)    -   1 μl ROX dye (50×, Invitrogen, Carlsbad, Calif.)    -   3.5 μl IsoAmpII enzyme mix (BioHelix, Beverly, Mass.)    -   1 μl SspI or H₂O (for the control without endonuclease)        add dH₂O to total 45 μl.        *10× Annealing buffer II contains 100 mM KCl and 200 mM Tris-HCl        (pH 8.8 at 25° C.)

For the test with plasmid DNA, in 4 individual PCR reaction tubes, 5 piof 10,000 copies/μ1 pBRCA-TOPO was added to each tube. 45 μl of reactionmaster mix with SspI was added to 2 of the tubes, and 45 μl of reactionmaster mix without SspI was added to the other two tubes. The reactionswere mixed by pipetting up and down several times and then covered withmineral oil. The reaction mixture was immediately incubated in anABI7300 real-time PCR machine with the well inspector setting: reporterdye: SYBR; quencher: none; passive reference dye: ROX. An ABI7300real-time PCR instrument was used to monitor the HDA reaction inreal-time because no isothermal real-time fluorescent instrument wascommercially available. To perform an isothermal reaction on an ABI7300real-time PCR instrument, which requires a minimum of 1 degree oftemperature variation between different cycles, the following programwas used:

Stage 1: (30×)

Step 1: 64.5° C. for 0:05

Step 2: 63.5° C. for 1:55

Data collection and real-time analysis enabled

Stage 2: (1×)

Dissociation Stage

Melt curve data collection and analysis enabled

For the test with human genomics DNA, a similar procedure was performed.The only difference was that in 4 individual PCR reaction tubes, 5 μl of4 ng/μ1 human genomic DNA (Promega, Madison, Wis.) were added to eachtube.

The graphs in FIG. 1 and FIG. 2 were generated by the software of theABI7300 realtime PCR System as log view of delta Rn versus cycle. When0.38-pg of plasmid DNA were used in the reactions (FIG. 1), the Ctnumber was reduced by about 2.5 cycles from around Ct 14 to 11.5(corresponding to a reduction of amplification time by 5 minutes) in thereaction with SspI compared to the reaction without SspI. However, when20 ng of human genomic DNA were used in the tests (FIG. 2), there was noobvious difference comparing the reactions with or without SspI. Theseresults demonstrate that in the presence of a high amount of DNA (withan overwhelming number of ends for helicase loading), no significantenhancement to the speed of the HDA reaction was observed.

Example 2 Use of Endonucleases to Improve Detection Sensitivity of HDAwhen the Target Copy Number is Low

In this example, endonuclease cleavage was used to improve thesensitivity of HDA detection. The same target (human genomic DNA) andprimers were used as in Example 1 and the same components of thereaction master mix were prepared.

For the assay with SspI endonuclease, 12× reaction master mix with SspIwas prepared for 10 samples (same components as described in Example 1).For the assay without SspI, 12× reaction master mix without SspI wasprepared for 10 samples. In 20 individual PCR reaction tubes, 5 μl ofthe following samples were added to each tube:

1-2: 0.2 ng/μl human genomic DNA (around 300 copies per reaction);

3-8: 2 pg/μl human genomic DNA (around 3 copies per reaction);

9-10: H₂O (negative control);

11-12: 0.2 ng/μl human genomic DNA (around 300 copies per reaction);

13-18: 2 pg/μl human genomic DNA (around 3 copies per reaction);

19-20: H₂O (negative control).

Then, 45 μl of reaction master mix with SspI were added to tubes 1-10,and 45 μl of reaction master mix without SspI were added to tubes 11-20.The reactions were mixed by pipetting up and down several times and thencovered with mineral oil. The reaction mixture was immediately incubatedin an ABI7300 real-time PCR machine with the same program as inExample 1. The results are given in Table 1 below.

TABLE 1 Comparison of Sspl effects from the amplification of low copynumber of human genomic DNA. Ct from 1 ng DNA Ct from 0.01 ng DNASuccess rate from (300 copies) (3 copies) 0.01 ng DNA +Sspl 14.62 ± 0.0818.36 ± 1.08   100% (6/6 positive) −Sspl 16.16 ± 0.04 22.87 16.67% (2/6fail, 3/6 dimmer)

For a medium copy number of targets (300 copies of human genomic DNA),the Ct was decreased by about 1.5 cycles (from Ct 16.1 to Ct 14.5) bythe endonuclease. For a low copy number of targets (3 copies of humangenomic DNA), without the help of SspI, only 1 out of 6 reactions wassuccessful (success rate around 17%). Two reactions failed and 3reactions generated primer dimers. However, with the help of SspI, thesuccess rate was 100% (6 out of 6 worked), with a standard deviation ofCt of about 1 Ct. This demonstrates that the robustness and sensitivityof the assay has been significantly improved by endonuclease cleavage.

Example 3 Use of Endonucleases Generating a 5′ End or a 3′ End toImprove the Reaction Speed of HDA

In this example, a method is disclosed to improve HDA reaction speed byendonucleases digestion which can generate 5′ ends or 3′ ends. The sameplasmid target pBRCA-TOPO was used as in Example 1 (50,000 copies foreach reaction). The target sequence of SpeI is A^(↓)CTAGT; the closestsite to the amplification region is around 66 bp upstream of the primerbinding site. Upon cleavage, 5′ ends are generated. The target sequenceof BanII is GRGCY^(↓)C (R represents A or G, Y represents C or T), theclosest site to the amplification region is around 387 bp upstream ofthe primer binding site. Upon cleavage, 3′ ends are generated.

The similar reaction master mix was prepared as Example 1. 4× reactionmaster mix were prepared for 2 tested samples (45μ/reaction):

-   -   5 μl 10× Annealing buffer II (see below*)    -   0.75 μl 5.0 μM BRCA2F primer (SEQ ID NO:1)    -   0.75 μl 5.0 μM BRCA2R primer (SEQ ID NO:2)    -   2.0 μl 100 mM MgSO₄    -   2.0 μl 500 mM NaCl    -   2.0 μl 10 mM dNTP    -   1.5 μl 100 mM dATP    -   0.5 μl EvaGreen (20×, Biotium, Hayward, Calif.)    -   1 μl ROX dye (50×, Invitrogen, Carlsbad, Calif.)    -   3.5 μl IsoAmpII enzyme mix (BioHelix, Beverly, Mass.)    -   1 μl SpeI, or BanII or H₂O (for the control without        endonuclease)        add dH₂O to total 45 μl.

For the test with SpeI, in 4 individual PCR reaction tubes, 5 μl of10,000 copies/μl pBRCA-TOPO were added to each tube. 45 μl of reactionmaster mix with SpeI were added to 2 of the tubes, and 45 μl of reactionmaster mix without SpeI were added to 2 of the other tubes. Thereactions were mixed well by pipetting up and down several times andthen covered with mineral oil. The reaction mixture was immediatelyincubated in an ABI7300 real-time PCR machine with the same program asExample 1.

A similar procedure was performed with BanII.

The graphs in FIG. 3 and FIG. 4 were generated by the software of theABI7300 realtime PCR System. The Ct was reduced by about 2 cycles(corresponding to 4 minutes, from around Ct 14 to 12) in the reactionwith SpeI compared to the reaction without SpeI. The Ct was also reducedby about 2 cycles (corresponding to 4 minutes, from around Ct 13 to 11)in the reaction with BanII compared to the reaction without BanII. (Thecorresponding melt cure analysis is not shown here.) This demonstratesthat the HDA reaction speed can be improved by the endonucleases thatcan generate either 5′ ends or 3′ ends, or blunt ends as Example 1.

It will be appreciated that several of the above-disclosed and otherfeatures and functions, or alternatives thereof, may be desirablycombined into many other different systems or applications.

We claim:
 1. A composition for amplifying a target nucleic acid sequencein a sample, comprising a type II restriction endonuclease, a helicase,a DNA polymerase, a DNA molecule comprising the target nucleic acidsequence, and at least a first primer and a second primer, wherein theDNA molecule is bound by the type II restriction endonuclease, thehelicase, the DNA polymerase and the at least first and second primers;wherein the sample may or may not contain DNA which comprises the targetnucleic acid; wherein the target nucleic acid is one which canhybridizes to at least the first and second primer and is amplified inthe presence of the composition to generate an amplification productwherein the 5′ end of the amplification product is defined by the 5′ endof the first primer and the 3′ end of the amplification product isdefined by the 5′ end of the second primer; wherein the type IIrestriction endonuclease is one which does not recognize or cleave theamplification product; and wherein the type II restriction endonucleaseis one which cleaves the target DNA within 5000 bases of the targetnucleic acid.
 2. The composition of claim 1, wherein the restrictionendonuclease is a Type IIs restriction endonuclease.
 3. The compositionof claim 1, wherein the restriction endonuclease is AvaII, BanII, AseI,SspI, SpeI, MboI, MboII, SwaI, BsrI, BtsCI, CviQI, CviAII, Tsp45I,Fnu4HI, MsII, MspAII, SacII, AgeI, DraI, or XmnI.
 4. The composition ofclaim 1, wherein the helicase is a thermostable helicase.
 5. Thecomposition of claim 1, wherein the composition further comprisesdeoxynucleotides.
 6. The composition of claim 1, wherein the compositionfurther comprises a buffer.
 7. The composition of claim 1, wherein thecomposition further comprises magnesium.
 8. A kit for amplifying atarget nucleic acid sequence in a sample, comprising a type IIrestriction endonuclease, a helicase, a DNA polymerase, a DNA moleculecomprising the target nucleic acid sequence, and at least a first primerand a second primer, wherein the DNA molecule is bound by the at leasttype II restriction endonuclease, the helicase, the DNA polymerase andthe first and second primers wherein the sample may or may not containDNA which comprises the target nucleic acid; wherein the target nucleicacid is one which can hybridize to at least the first and second primerand is amplified in the presence of the composition to generate anamplification product wherein the 5′ end of the amplification product isdefined by the 5′ end of the first primer and the 3′ end of theamplification product is defined by the 5′ end of the second primer;wherein the type II restriction endonuclease is one which cannotrecognize or cleave the amplification product; and wherein the type IIrestriction endonuclease is one which cleaves the target DNA within 5000bases of the target nucleic acid.
 9. The kit of claim 8, wherein therestriction endonuclease is a Type IIs restriction endonuclease.
 10. Thekit of claim 8, wherein the restriction endonuclease is AvaII, BanII,AseI, SspI, SpeI, MboI, MboII, SwaI, BsrI, BtsCI, CviQI, CviAII, Tsp45I,Fnu4HI, MsII, MspAII, SacII, AgeI, DraI, or XmnI.
 11. The kit of claim8, wherein the helicase is a thermostable helicase.
 12. The kit of claim8, wherein the kit further comprises deoxynucleotides.
 13. The kit ofclaim 8, wherein the kit further comprises a buffer.
 14. The kit ofclaim 8, wherein the kit further comprises magnesium.
 15. Thecomposition of claim 1, wherein the type II restriction endonuclease isone which can recognize and cleave the DNA within 500 bases of thetarget nucleic acid.
 16. The composition of claim 8, wherein the type IIrestriction endonuclease is one which can recognize and cleave the DNAwithin 500 bases of the target nucleic acid.